We constructed a hybrid strain that acquired 3,4-dioxygenase activity for polychlorinated biphenyls (PCBs). This strain, KF707-D34, possessed a chimeric biphenyl dioxygenase gene, of which a portion of bphAl (coding for a large subunit of biphenyl dioxygenase) of Pseudomonas pseudoalcaligenes KF707 was replaced with that of a PCB-degrader, Burkholderia cepacia LB400 by homologous recombination. KF707-D34 retained the ability to degrade 4,4'-dichlorobiphenyl via 2,3-dioxygenation in a fashion identical to that of KF707 and gained novel capability to degrade 2,5,4'-trichlorobiphenyl and 2,5,2,5'-tetrachlorobiphenyl via 3,4-dioxygenation in a fashion identical to that of LB400. Sequence analysis of bphAl from KF707-D34 revealed that three nucleotides in the 3'-terminal region of KF707 bphAl were changed to correspond to those in LB400 bphAl. The resulting BphAl protein in KF707-D34 was changed at position 376 from threonine (Thr) to asparagine (Asn). The results demonstrate that a minor alteration of the amino acid sequence in BphAl improved the PCB degradation capability in biphenyl-utilizing bacteria.